Fetal thymic organ culture of TCR transgenic (Tg) tissue has been used to study issues of timing and specificity in T cell development. Because most TCR Tgs express a rearranged alphabeta TCR on the cell surface at an earlier stage in development than normal mice, there is a possibility that the conclusions of studies using TCR Tg cultures may not apply to normal development. In particular, in our studies of peptide-induced development of CD8 T cells, it is possible that the peptide acts on the immature double-negative cell, driving development of CD8 T cells without passing through a double-positive stage. This issue was examined by asking whether MHC class I restriction was required and by analyzing CD8beta levels and endogenous TCR alpha chain rearrangements. We found that if nonstimulatory peptides were used in fetal thymic organ culture, CD8 T cells developed via the conventional pathway, transiting through a double-positive stage. However, we could not rule out that cells selected in the presence of stimulatory peptides (agonists) did not develop directly from double-negative precursors.